The present invention relates in general to radioimmuno conjugates and in particular to radioimmuno conjugates of the type to be used in the therapy of human patients for the treatment of malignant haematopoietic diseases. The invention relates furthermore to a method for preparing the radioimmuno conjugates.
In modern therapy, two types of treatments are normally applied with malignant haematopoietic diseases: In one type of treatment, high doses of differentiated chemotherapy is followed by bone marrow and/or stem cell transplantation. In the other type of treatment, high doses of differentiated chemotherapy and whole body irradiation is followed by bone marrow and/or stem cell transplantation. The goal of the aggressive, high-dose and mostly differentiated chemotherapy or radiation of the full body which is carried out with energy rich, hard gamma rays (e.g. high voltage therapy with a .sup.60 Co-Cobalt-source), or a combination of the two therapies, is to kill the malignant cells as well as their precursor cells generated in surplus in the bone marrow. This is preferably done in a quantitative or semi-quantitative manner already at the level of their common pluripotent stem cells. This procedure also causes the destruction of the blood forming-, granulocyte producing--as well as the thrombocyte forming--system in the bone marrow and without any further therapeutic measures, this strong immuno suppression would lead unequivocally to the patient's death. Only immediate intravenous injection of bone marrow suspensions and/or suspensions of allogenic stem cells or, whenever possible, suspensions of autologous bone marrow and purified bone marrow obtained through purging techniques, guarantees the survival of the patient. The cells which are intravenously supplied, subsequently settle in the lymphoid organs such as the bone marrow and the spleen. A few weeks after the patient has undergone chemotherapy and/or full body radiation during which the patient's haematopoiesis was destroyed, these cells, by virtue of undergoing mitosis, differentiation and maturing, develop into fully functioning blood components such as erythrocytes, granulocytes, thrombocytes, myelocytes, monocytes. Administration of these intensive therapies has resulted in a markedly improved survival rate of those patients suffering from various malignant haematopoietic diseases.
Such therapies have however, various shortcomings. In addition to the destruction of haematopoisis and the serious changes induced by the immuno suppression, there are also additional side effects caused by the highly concentrated chemotherapy and/or whole body radiation which causes further damage to other essential organs. Such organ damage manifests itself for example in hair loss, nausea, vomiting and general malaise of the patient.
In addition to such observed side effects as kidney-toxicity (Mirabell et al. J. Clin. Oncol. 1996, 14 (2) 579-585), cataracts (Alevard T. Acta Oncol. 1996, 35 (7) 137-140), germ cell dysfunction (Sarafoglou et al. J. Pediatr. 1997, 130 (2) 210-216) which occur with high frequency, there were also cases where acute respiratory system threatening noninfectious epiglottitis has occurred (Murray et al. Bone Marrow Transplantation 1995, 15 (6) 997-998).
Numerous patients with malignant haematopoietic diseases are children making it thus an even more urgent medical need to improve the treatments so there are fewer side effects and also in particular, to improve their efficacy.
Within the last fifteen years, a number of experiments have been carried out using unlabeled monoclonal antibodies (MAb) and/or radio-labeled monoclonal antibodies (radio immune conjugates) in order to improve the treatment of malignant diseases. It was only within the past five years that the clinical efficacy of unlabelled cytotoxic MAb could be shown. Thus, Riethmuller et al. (Lancet 1994, 343 1177-1183) could show ascertainable statistics in life prolongation with a cytotoxic, non genetically-engineered MAb from a mouse used in a randomized clinical study during the treatment of "minimal residual disease" in colon carcinoma.
So far, the clinical efficacy of unlabelled MAb has not yet been shown in the treatment of larger tumor masses, even though, in addition to the MAb from the mouse, humanized and genetically engineered MAbs and their fragments have also been used. (Colnaghi et al. Current Opinion in Oncology 1993, 5, 1035-1042). For this reason, a number of researchers have tried to radio label MAb as well as their gene-technological variants of different specificity, origin and size, such as for example cytostatic agents or radioactive isotopes (Courtenay-Luck and Epenetos, Immunology 1990, 2 880-883). In order to bind the toxic components at the MAb, divers coupling and labeling methods were used.
Only in the past several years, suitable radionuclides with advantageous radiation properties, such as for example, Phosphorus-32, Strontium-89, Yttrium-90, Samarium-153, Erbium-169, Ytterbium-175, Rhenium-188 were successfully and stably coupled to MAb for therapeutic purposes by means of bi-functional complex forming agents (while iodine has been known for many years to couple to MAb, it is not particularly useful because of its unfavorable radiation properties).
Radiochemical methods for labelling MAb using various radionuclides are divided into two groups: the direct labeling methods and the indirect labeling methods. With the direct method, the inner di-sulfide bonds (--S--S--) of the hinge-region of the MAb are being partially reduced to sulfhydril groups (--SH). To do this, various chemical compounds having reducing properties are used, such as for example, the derivatives of ascorbic acid (Hnatowich D. et al. J. Nucl. Med. 1994; 35: 127-134), and/or substances with sulfhydryl groups or stannic-II-compounds or complexes (Mather S. et al. J. Nucl. Med. 1990; 31: 692-697, Paik C et al J. Nucl. Med. Biol. 1985; 12: 3-8, Rhodes B. J. Nucl. Med. 1986: 27: 685-693, Thakur M. et al. Nucl. Med. Biol. 1991; 18: 227-233, Schearz A. et al. J. Nucl. med. Biol. 1986: 28: 721). Experiments to couple Re-186 or Re-188 stably onto a MAb without using complex forming agents, have led to the formation of conjugates, according to the present level of knowledge (Visser et al. J. Nucl. Med. 1993, 3, 1953-1963, see page 1962, lines 34-38) that are not sufficiently stable (Griffiths et al. Cancer Res. 1991, 51, 4594-4602, Su et al. J. Nucl. Med. 1992, 33, 910).
In the indirect labeling methods of MAb, mostly bi-functional complex forming agents are used, such as diamine-dithiol (Baidoo K. et al. Cancer Res. 1990: 50: 799-803), or a bi-functional ester of NHS-BAT (Eisenhut M. et al. J Nucl Med 1991; 37: 362-370), or diamid-dimercaptid (Kasina S. et al. J. Nucl. Med. 1991; 32: 1445-1451) and/or DTPA (Najafi A. et al. Int. J. Appl. Radiat. Isot. 1984; 5: 554-557), or a novel complex forming agent, which is based upon a N2S4-composition (Najafi A. et al. Nucl. Med. Biol. 1991; 18: 179-185, Qu T. et al. Radiochim. Acta 1993; 63: 209-212) for conjugating the radionuclide onto the MAb. Alternative methods which facilitate an indirect coupling (conjugation) of the radionuclide to the MAb are based on the conjugation of thiol-groups to amino acids (e.g. lysine) in the protein molecule with 2-iminothiolan (Joiris E. et al. Nucl. Med. Biol. 1991, 18: 353-356) or with the groups of 1-imino-4-mercaptobutyl compounds (Goedemans W. in Nicolin M. et al. (eds.) Verona 1990; 595-603).
Suitable complex forming agents for the complex formation, especially with Yttrium (preferably Y-90) are, for example, DOTA (Denora et al. Anticancer Research 1997, 17, 1735-1744) or 12 N4-maleimid (tetra-azocyclododecantextra-acetic acid) (Turner et al. Br. J. Cancer, 1994, 70: 35-41, and King et al. Cancer Research, 1994, 54: 6176-6185). Particularly suited for complex formation of Rhenium (preferably Re-186 or Re-188), is for example, the MAG-3 complex forming agent (van Gog et al. J. Nucl. Med. 1996, 37, (2), 352-362). These methods suffer, however, from the disadvantage that an immuno response may be formed against the complex-forming portion of the MAb-complex forming conjugate. Thus, none of the prior known methods have produced a satisfactory conjugate for the treatment of malignant haematopoietic diseases. Furthermore, the available therapies for treating the afore-mentioned highly malignant haematopoietic diseases is unsatisfactory due to the massive side effects they provoke. It was therefore an object of the inventors to develop a novel radioimmuno conjugate for use in treating these diseases which by its application would replace, or at a minimum reduce the need for numerous applications of high dose chemotherapy or total body irradiation.